The long-term objectives of the proposed research are to identify factors that play an important role in transcription initiation by RNA polymerase II in vivo and to determine the mechanisms by which these factors govern this process. The initial focus of these studies will be the TATA box- binding general transcription factor (TATA box-binding protein or TBP). At promoters transcribed by RNA polymerase II, binding of TBP to the TATA box initiates the ordered assembly of a multi-protein preinitiation complex that is sufficient to support basal levels of transcription initiation in vitro. Additional proteins are required for the response to regulatory signals. Since TATA box binding by TBP is the first step in preinitiation complex assembly, TBP is an important target for the action of regulatory factors. The Specific Aims of this proposal are to investigate three different aspects of TBP function: (l) the role of TBP as a target for transcriptional activators; (2) the determinants of sequence-specific and orientation-specific DNA binding by TBP; and (3) the functions of two structural domains of TBP that are likely targets for interacting proteins. To accomplish these aims, a combination of genetic and biochemical approaches will be employed to identify and characterize proteins that interact with and regulate the activity of TBP in the yeast Saccharomyces cerevisiae. More specifically, genetic selections will be performed to identify mutations that suppress or enhance the phenotypes conferred by TBP mutants. The foundations for these studies are different classes of TBP mutants, both previously identified mutants and new mutants that will be isolated during the course of the proposed research. One existing class of TBP mutants exhibits defects in activated transcription in vivo, and a second class exhibits alterations in DNA-binding specificity. Unlinked mutations that alter the phenotypes caused by these TBP mutants will be characterized, and corresponding genes will be cloned and studied by genetic and molecular approaches. Subsequently, to elucidate the mechanisms by which the products of these genes alter TBP function, proteins will be purified and analyzed by biochemical methods that will include in vitro transcription and DNA-binding assays. Since the proteins and mechanisms employed in transcription initiation by RNA polymerase II have been highly conserved throughout evolution, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in other eukaryotes. Thus, the proposed research is expected to provide valuable insights into the regulation of transcription initiation in humans where a genetic alteration in this process can disrupt normal cell growth and differentiation, leading to cancer and other diseases.